PROJECT SUMMARY Here, we will generate photoswitchable and photoactivatable bioluminescent (PS-BL and PA-BL) light sources. These constructs will be implemented to facilitate highly selective and ?reprogrammable? modulation of neural ensembles. The PS-/PA-BL approach allows rapid selection (minutes), quick re-set (hours), and can be implemented across depths by selective 2-Photon activation. Neural control will be implemented by using PS-BL light to drive optogenetics (PS-BL-OG), a powerful modulation strategy. The PS-/PA-BL approach exceeds efficacy and spatial extent of current simultaneous optogenetic regulation by holography. The PS-/PA- BL approach also has distinct advantages over methods expressing optogenetic sensitivity in cells based on activity-linked transcription by faster on- and off-set and by giving the experimenter control over selection of cells by criteria other than that of highest activity. We will test two independent mechanisms for achieving light-based modulation of bioluminescence emission. In Aim I, we will generate PS-BL by fusing luciferases to reversibly photoswitchable fluorescent proteins, selecting for optimal Frster resonance energy transfer and bright emission following optical activation. In Aim II, we will generate PA-BL by fusing luciferases to irreversibly photoactivatable and photoconvertible fluorescent proteins. Directed evolution of these constructs will improve on-off contrast, 2P switching efficiency, and switching kinetics. The most effective PS-/PA-BL constructs will then be combined with optogenetic elements to generate PS-/PA-BL-OG probes for targeted chemogenetic control of selected neurons. Both Aims will use an iterative process of probe design and engineering, directed evolution with image-based screening, and testing for light production and neural control ex vivo, in vitro and in vivo in mouse neocortex to determine the specific optical and biochemical properties requiring further optimization. End products will be validated BL probes that provide new strategies for neural control. The team has the expertise across levels to bring this goal from molecular engineering to in vivo realization.